I am currently trying to do a CRISPR/Cas9 knock-in and am wondering about the design of the donor vector (with the HDR template).
I've been looking through many publications, but never actually find an explanation how the backbone is chosen. If the vector consists of an ORI and an antibiotic selection marker, can you just take any backbone, like pUC19 or so on, or does a knock-in require certain plasmid designs (apart from the insertion cassette, of course)?
Thank you!
Since you are not wanting to keep the backbone as an extrachromosomal element you don't care if the origin works in the destination species. Yes you want a bacterial antibiotic selection marker for convenience when cloning. Yes you ideally want a selective marker for your target species in your insertion cassette. The main factor for choosing which backbone to use is a small size to increase transfection efficiency and the restriction sites in the MCS which must be compatible with restriction sites in the homology arms. Adding a single strand annealing protein expression cassette outside your insertion cassette can increase homologous recombination frequency, improving the likelihood your knock in occurs. This means you can use pUC backbones, pBluescript or whatever you prefer.
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